Interleukin-1 (IL-1) is a polypeptide cytokine of 17.5 kDa. Two forms of IL-1 (IL-1.alpha. and IL-1.beta.) are released from a variety of cells in response to inflammatory insult. IL-1.alpha. and IL-1.beta. are a part of a complex network involved in mediating a wide range of immune responses. Dinarello, Eur. Cytokine Netw., 5, 517 (1994); Dinarello, The FASEB Journal, 8, 1314 (1994); Sims and Dower, Eur. Cytokine Netw., 5, 539 (1994). IL-1 exerts pleiotropic effects on host defenses via two kinds of receptor proteins. The larger protein, type I (80 kDa), is responsible for transducing IL-1 signals across the cell membrane, whereas the smaller protein, type II (68 kDa), functions as an apparent "decoy" protein which binds IL-1 but does not transduce a pro-inflammatory intracellular signal. Colota et al., Science, 261, 472 (1993); Sims et al., Clin. Immunol. Immunopath., 72, 9 (1994); Sims and Dower, Eur. Cytokine Netw., 5, 539 (1994). An additional member of the IL-1 receptor (IL-1r) family is the IL-1 receptor antagonist (IL-1Ra), which is a specific soluble antagonist of both IL-1.alpha. and IL-1.beta.. IL-1Ra binds to both the IL-1 receptors (types I and II) with affinities similar to that with which it binds to IL-1.alpha. and IL-1.beta., but does not transduce a signal. Arend, Adv. Immunol., 54, 167 (1993); Dayer and Burger, Eur. Cytokine Netw., 5, 563 (1994).
The normal function of IL-1 is as an inflammatory cytokine producing multiple effects in the body. Although these functions are important in response to infection and injury, IL-1 expression and secretion can be deleterious when occurring at high concentrations or for extended periods of time. Sims et al., Clin. Immunol. Immunopath., 72, 9 (1994); Dinarello, Eur. Cytokine Netw., 5, 517 (1994); Dinarello, Blood Purif., 11, 118 (1993); Dinarello and Wolff, N. Engl. J. Med., 328, 106 (1993).
A number of different strategies have been employed in an attempt to inhibit IL-1 receptor-mediated effects in order to examine the specific role played by IL-1 in various disease processes. These include administration of antibodies targeting either the IL-1 receptor Geiger et al., Clin Exp. Rheumatol., 11, 515 (1993); McNamara et al., J. Surgical Res., 54, 316 (1993)! or IL-1Ra Arend, Adv. Immunol., 54, 167 (1993); Dinarello and Thompson, Immunol. Today, 12, 404 (1991); Schotanus et al., Endocrinology, 133, 2461 (1994)!.
Another approach to inhibiting the expression of IL-1r is by employing antisense oligonucleotides. Most conventional therapeutic agents exert their effect by interaction with and modulation of one or more targeted endogenous proteins. If this modulation could be effected by interaction with DNA and/or subsequent inhibition of mRNA expression, a dramatic reduction in the amount of therapeutic agent required to effect inhibition of the activity of the protein could be achieved. As this approach allows highly selective targeting of particular DNA or RNA sequences, another advantage of antisense oligonucleotides as inhibitors of protein expression is the specificity which may be achieved accompanied by a corresponding decrease in side effects resulting from such treatment.
However, some oligonucleotides are susceptible to enzymatic degradation by a variety of ubiquitous nucleases which may be intracellularly or extracellularly located. The efficacy of unmodified, "wild-type" oligonucleotides (i.e., those containing phosphodiester linkages and 2'-deoxy-erythro-pentofuranosyl sugar moieties) as therapeutic agents may be suboptimal because they are rapidly degraded by nucleases. Therefore, modification of oligonucleotides for conferring nuclease resistance on them has been a focus of research directed towards the development of oligonucleotide therapeutics and diagnostics. In addition to nuclease stability, the ability of an oligonucleotide to bind to a specific DNA or RNA with fidelity is a further important factor.
Oligonucleotides modified to exhibit resistance to nucleases and to hybridize with appropriate strength and fidelity to target DNA or RNA are greatly desired for use as research reagents, diagnostic agents and as oligonucleotide therapeutics. Various 2'-substitutions have been introduced in the sugar moiety of oligonucleotides. The nuclease resistance of these compounds has been increased by the introduction of 2'-substituents such as halo, alkoxy and allyloxy groups.
Ikehara et al. Eur. J. Biochem., 139, 447 (1984) have reported the synthesis of a mixed octamer containing one 2'-deoxy-2'-fluoroguanosine residue or one 2'-deoxy-2'-fluoroadenine residue. Guschlbauer and Jankowski Nucleic Acids Res., 8, 1421 (1980) have shown that the contribution of the 3'-endo increases with increasing electronegativity of the 2'-substituent. Thus, 2'-deoxy-2'-fluorouridine contains 85% of the C3'-endo conformer.
Ikehara et al. Nucleic Acids Res., 4, 4249 (1978) have shown that a 2'-chloro or bromo substituent in poly(2'-deoxyadenylic acid) provides nuclease resistance. Eckstein et al. Biochemistry, 11, 4336 (1972) have reported that poly(2'-chloro-2'-deoxyuridylic acid) and poly(2'-chloro-2'-deoxycytidylic acid) are resistant to various nucleases. Inoue et al. have described the synthesis of mixed oligonucleotide sequences containing 2'-O-methyl substituents. The mixed 2'-O-methyl-substituted oligonucleotide hybridized to its RNA complement as strongly as the RNA-RNA duplex which is significantly stronger than the same sequence RNA-DNA heteroduplex (T.sub.m s, 49.0 and 50.1 versus 33.0 degrees for nonamers). Nucleic Acids Research, 15, 6131 (1987). Shibahara et al. have reported the synthesis of mixed oligonucleotides containing 2'-O-methyl substituents which were designed to inhibit HIV replication. Nucleic Acids Research, 17, 239 (1987).
U.S. Pat. 5,013,830, issued May 7, 1991, discloses mixed oligonucleotides comprising an RNA portion, bearing 2'-O-alkyl substituents, conjugated to a DNA portion via a phosphodiester linkage. However, being phosphodiesters, these oligonucleotides are susceptible to nuclease cleavage.
European Patent application 339,842, filed Apr. 13, 1989, discloses 2'-O-substituted phosphorothioate oligonucleotides, including 2'-O-methylribooligonucleotide phosphorothioate derivatives. This application also discloses 2'-O-methyl phosphodiester oligonucleotides which lack nuclease resistance.
International Publication Number WO 91/06556, published May 16, 1991, discloses oligomers derivatized at the 2' position with substituents, which are stable to nuclease activity. Specific 2'-O-substituents which were incorporated into oligonucleotides include ethoxycarbonylmethyl (ester form), and its acid, amide and substituted amide forms.
European Patent application 399,330, filed May 15, 1990, discloses nucleotides having 2'-O-alkyl substituents.
International Publication Number WO 91/15499, published Oct. 17, 1991, discloses oligonucleotides bearing 2'-O-alkyl, -alkenyl and -alkynyl substituents.
U.S. Pat. No. 5,135,917, issued Aug. 4, 1992, discloses oligonucleotide compounds having 2'-deoxy sugar moieties which bind to mRNA coding for human IL-1 receptors. The oligomers of the present invention differ from the compounds described in this patent in that the oligomers disclosed in the present application comprise nucleosides bearing 2'-substituents on the sugar moieties. The 2'-substitution on sugar moieties makes oligomers of the invention nuclease resistant.
It has been recognized that nuclease resistance of oligonucleotides and fidelity of hybridization are of great importance in the development of oligonucleotide therapeutics. Oligonucleotides possessing nuclease resistance are also desired as research reagents and diagnostic agents.